1. Field of the Invention
The present invention relates to the field of genetically engineered peptide production in plants, more specifically, to the use of tobamovirus vectors to express fusion proteins, and more specifically, to a vaccine comprising an encapsidated virus having a modified coat protein which displays a parvovirus antigen.
2. Description of the Background Art
Peptides are a diverse class of molecules having a variety of important chemical and biological properties. Some examples include; hormones, cytokines, immunoregulators, enzyme inhibitors, vaccine antigens, adhesion molecules, receptor binding domains, and the like. The cost of chemical synthesis limits the potential applications of synthetic peptides for many uses such as therapeutic drugs or vaccines. There is a need for inexpensive and rapid synthesis of milligram and larger quantities of naturally occurring polypeptides. Towards this goal many animal and bacterial viruses have been used successfully as peptide carriers.
The safe and inexpensive culture of plants provides an advantageous alternative for cost-effective production of pharmaceutically useful peptides. During the last decade, considerable progress has been made in expressing foreign genes in plants. Foreign proteins are now routinely produced in many plant species either for modification of the plant or for protein extraction and production. Animal proteins have been effectively produced in plants (reviewed in Krebbers, E. et al., In: Plant Protein Engineering (P. R. Shewry et al., eds.), Cambridge University Press, Cambridge, 1992, pp.316-324).
Vectors for the genetic manipulation of plants have been derived from several naturally occurring plant viruses, including tobacco mosaic virus (TMV). TMV is the type member of the tobamovirus group. TMV has straight tubular virions of approximately 300xc3x9718 nm with a 4 nm-diameter hollow canal, consisting of approximately 2000 units of a single capsid protein wound helically around a single RNA molecule. Virion particles are 95% protein and 5% RNA by weight. The genome of TMV is composed of a single-stranded RNA of 6395 nucleotides containing five large open reading frames (ORFs). Expression of each gene is regulated independently. The virion RNA serves as the messenger RNA (mRNA) for the 5xe2x80x2 genes, encoding the 126 kDa replicase subunit and the overlapping 183 kDa replicase subunit that is produced by read-through of a UAG stop codon approximately 5% of the time. Expression of the internal genes is controlled by different promoters on the minus-sense RNA that direct synthesis of 3xe2x80x2-coterminal subgenomic mRNAs which are produced during replication (FIG. 1). A detailed description of tobamovirus gene expression and life cycle can be found, among other places, in Dawson and Lehto, Adv. Vir. Res. 38:307-342 (1991). It is of interest to provide new and improved vectors for the genetic manipulation of plants.
For production of specific proteins, transient expression of foreign genes in plants using virus-based vectors has several advantages. Products of plant viruses are among the highest produced proteins in plants. Often a viral gene product is the major protein produced in plant cells during virus replication. Many viruses are able to spread quickly from an initial infection site to almost all cells of the plant. For these reasons, plant viruses have been developed into efficient transient expression vectors for foreign genes in plants. Viruses of multi-cellular plants are relatively small, probably due to the size limitation in the pathways that allow viruses to move to adjacent cells in systemic infection of the entire plant. Most plant viruses have single-stranded RNA genomes of less than 10 kb. Genetically altered plant viruses provide one efficient means of transfecting plants with genes encoding peptide-carrier fusion proteins. A discussion of TMV coat protein fusions is provided in Turpen et al., U.S. Pat. No. 5,977,438 entitled xe2x80x9cProduction of Peptides in Plants as Viral Coat Protein Fusions.xe2x80x9d Nov. 2, 1999. See also: Yusibov V. et al., Proc. Natl. Acad. Sci. USA 94:5784-5788 (1997); Modelska, A et al., Proc. Natl. Acad. Sci. USA 95:2481-2485 (1998).
The pathogenesis of parvovirus infection has been most recently reviewed by Parish, C. R., Baillieres Clin. Haematol. 8:57-71, (1995.). Feline parvovirus (FPV) is closely related to canine parvovirus and the respective diseases are similar in pathogenesis. Parvovirus replicates first in the tonsils, and then spreads to its target cells: mitotically active intestinal crypt epithelial cells and bone marrow stem cells. Viremia lasts for less than 7 days before death or recovery. Clinical signs in cats include fever, vomiting, diarrhea, panleukopenia, acute shock and death. The disease outcome is proportional to the severity of the leukopenia; cats with severe panleukopenia will often die, while those with mild leukopenia will usually survive.
The VP2 (or E2) epitope of mink enteritis virus (MEV), which is closely related to FPV, has been previously expressed on the surface of cowpea mosaic virus, which was propagated on the leaves of the black-eyed bean (Dalsgaard, K et al., Nature Biotechnol. 15:248-252 (1997)). One mg of the cow pea mosaic virus material that expressed this epitope was used to immunize minks against virulent MEV. The minks were protected against clinical disease, and shed very little virus. The authors suggested that this epitope, expressed in this manner, could also be used to protect cats and dogs against their respective parvovirus infections.
The coding sequence for VP2 (E2) and the rabies spike glycoprotein have also been engineered into raccoon poxvirus to make a five recombinant vaccine against rabies and feline panleukopenia (Hu, L. et al., 1996. Virology 218:248-252., Hu, L. et al., 1997, Vaccine 15: 1466-1472.). Cats vaccinated with this construct showed excellent protection against virulent parvovirus challenge.
Citation of the above documents is not intended as an admission that any of the foregoing is pertinent prior art. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicant and does not constitute any admission as to the correctness of the dates or contents of these documents.
The present invention provides recombinant plant viruses that express fusion proteins that are formed by fusion between a plant viral coat protein (VCP) and a peptide or polypeptide of interest, primarily a peptide that bears an epitope of FLV. By infecting plant cells with the recombinant plant viruses of the invention, relatively large quantities of the peptide, in the form of a fusion protein, is produced. The fusion protein encoded by the recombinant plant virus may be engineered to have a variety of structures. The peptide may be fused to the amino terminus (N-terminus) or to the carboxy-terminus (C-terminus) of the VCP. Alternatively, the peptide may be fused internally into a coat protein (so that it is flanked on either side by coat protein sequences) The VCP fusion protein should have one or more properties of the fused peptide/polypeptide. The recombinant coat fusion protein may be used as an immunogen or antigen to induce an antibody response and protective immunity, or as reagent for developing and conducting immunoassays.
This invention also provides a polynucleotide that includes the genome of a recombinant plant virus. In another aspect, the invention provides the coat fusion protein that is encoded by the recombinant plant virus. Yet another embodiment is a plant cell or a whole plant that has been infected with this recombinant plant virus.
In particular, the invention provides a polynucleotide encoding a fusion protein capable of being expressed in a plant or a plant cell, wherein the fusion protein comprises (a) a plant VCP from a single-stranded plus-sense RNA virus fused to a peptide of interest, preferably comprising the amino acid sequence MGSDGAVQPDGGQPAV [SEQ ID NO:1] or comprising the amino acid sequence MGQPDGGQPAVRNERAT [SEQ ID NO:2], and (b) a promoter functional in plants that is situated 5xe2x80x2 to the fusion protein coding region.
The present invention is an important step forward in the art as it provides:
1. Stable, soluble, extractable TMV N-terminal coat protein fusion products useful as immunogens and vaccines.
2. The first description of a polyethyleneimine based virus extraction method.
3. High yields and stabilities of protein product based upon the plant hosts that express, and extraction methods that are used to purify, the product. The interplay of the particular production host and extraction methods that maximize the amount of undegraded product contribute to the high yields.
4. Specific and safe immunologically active epitopes decorating virus particles.
The novel aspects of this invention are not limited in application to the disclosed parvovirus vaccines, but rather are applicable to the highly efficient and inexpensive production of any relevant biologically active protein product for commercial use. The present inventors have shown that TMV-based vectors can express small antigenic peptides on the virion surface, that these virions can be easily and highly purified from infected leafs, and that an exemplary peptide (FPV-E2) provides the basis for a safe and efficacious vaccine requiring no further adjuvant over and above the immunogen itself The interstitial fluid of tobacco plants infected with particular TMV vectors containing the appropriate gene is safe, without requiring further purification, for pharmaceutical use, for example for injection into cats as a parvovirus vaccine.